An asymmetry in the synthesis of leading and lagging DNA strands creates the “end problem” for replication of linear genomes8. To overcome this, eukaryotic chromosomes have specialized end structures, telomeres, consisting of TTAGGG repeats9. Telomerase is a ribonucleoprotein enzyme that elongates telomeres and therefore maintains chromosomal stability in majority of cancer cells during cell doubling. The gradual loss of DNA from the ends of telomeres during cell doubling has been implicated in the control of cellular proliferative potential in somatic cells10.
Normal cultured human cells have a limited replication potential in culture. As was first described by Hayflick, normal cells in culture replicate until they reach a discrete point which population growth ceases. This is termed M1 stage and is caused by the shortening of a few telomeres to a size that leads to a growth arrest called cellular senescence. This stage can be bypassed in vitro by abrogation of the function of p53 and pRB human tumor suppressor genes. The cells then can proliferate until the telomeres have become critically shortened, which produces the M2 or crisis stage. The growth arrest in the M2 stage is caused by balance between the cell proliferation and cell death rate. At this stage, when most of telomeres are extremely short, end-to-end fusions and chromosomal breakage-fusion cause marked chromosomal abnormalities and apoptosis. Under rare circumstances, a cell can escape M2 and become immortal by stabilizing the length of its telomeres. This occurs through the activation of the enzyme telomerase or an alternative mechanism of telomere lengthening (ALT).
Human germline2 and the majority of cancer cells3 express telomerase. Telomerase is a ribonucleoprotein enzyme that elongates telomeres and, therefore, maintains chromosomal stability in majority of cancer cells during cell doubling. Indeed, elongation of shortened telomeres by telomerase is a major mechanism of telomere maintenance in the human cancer cells. Inhibition of telomerase limits the growth of human telomerase positive cancer cells11 by decreasing telomere length, these compounds diminish the ability of these cancer cells to proliferate. Reverse transcriptase inhibitors have been used previously to treat cancer. In in vitro tests, tumor cells treated with the reverse transcriptase inhibitors underwent apoptosis after 14 days.
Elongation of shortened telomeres by telomerase is a well known mechanism of telomere maintenance in the human cancer cells. However up to 30% of human tumors of different types do not express telomerase. The presence of ALT was reported in up to 30% of human tumors of different types, tumor-derived cell lines and human cell lines immortalized in vitro4,5,12,13, and up to 50% in some subsets of tumors and immortalized cell lines14.
Currently, strategies aimed at selectively treating the cancers from telomerase positive cells involve modulation of TERT function or length of telomeres by antisense strategy, dominant negative mutants or pharmacological agents (see, Bisoffi et al., Eur J Cancer, 1998, 34:1242-1249; Roth et al., Leukemia, 2003, 17:2410-2417; Damm et al., EMBO J., 2001, 20:6958-6968; U.S. Pat. Nos. 6,294,332, 6,194,206, 6,156,763 and 6,046,307). Selective modulation (i.e., selective inhibition or promotion) of telomerase negative cancer cells may also be made possible if the target molecule(s) responsible for the lengthening of telomeres in such cells are known. Thus, there is need for identifying target molecules responsible for the lengthening of telomeres in telomerase negative cells and identifying agents for selectively interfering with the identified target molecules so that human tumors of types that do not express telomerase may also be prevented or treated.